Cells that survive acute murine SARS-CoV-2 infection are detected nearly exclusively in the respiratory tract
Authors: Ruangang Pan, David K. Meyerholz, Stanley Perlman
Journal: Journal of Clinical Investigation (2023)

To the Editor: Long-term sequelae of SARS-CoV-2 infection, referred to as postacute sequelae of COVID-19 (PASC), involve many organs, including the cardiovascular, pulmonary, gastrointestinal, and neurological systems.The basis of PASC is not well understood, but one possible explanation is that the persistence of infectious virus or viral RNA or protein contributes to PASC.This is difficult to assess in patients because human tissue can only usually be assessed for virus or viral products at autopsy or by biopsy.Furthermore, it is not known if some SARS-CoV-2-infected cells survive the acute infection and contribute to long-term sequelae.To address this issue in mice, we used a lineage-tracing approach that allows longitudinal tracking of previously infected cells over several months (1, 2).For this purpose, two recombinant SARS-CoV-2 expressing cre recombinase and Venus fluorescent proteins were engineered using a previously described BAC reverse genetics system (3) (see Supplemental Methods; supplemental material available online with this article; https://doi.org/10.1172/JCI172659DS1).We used these viruses to infect mice expressing a loxP-flanked STOP cassette in the tdTomato gene driven by the Rosa26 promoter (Ai9 mice).Venus and Cre were linked by a 2A peptide (Venus-2a-Cre [V2C]), which after autocleavage resulted in the release of the two proteins (Figure 1A).V2C was inserted into ancestral SARS-CoV-2 (Wuhan-Hu-1; rSARS2-WH-V2C), which cannot infect laboratory mice but can infect hACE2 transgenic (K18-hACE2) mice (4), and into mouse-adapted SARS2-N501Y MA30 (rSARS2-MA30-V2C), which infects all strains of laboratory mice (5).Venus facilitated identification of acutely infected cells (Figure 1, B-D), while Cre expression resulted in permanent tdTomato labeling of cells that survive the acute infection (Figure 1, B andC).rSARS2-WH-V2C and rSARS2-MA30-V2C were as virulent as control viruses in K18-hACE2/Ai9 and Ai9 mice, respectively (Figure 1E and Supplemental Table 1).Pathological changes in the lungs (degree of hemorrhage, edema, perivascular infiltrates) were consistent with virulence (Figure 1F and Supplemental Methods).Venus expression was detected between 1 and 5 days after infection (dpi), with peak expression found at 2 dpi.tdTomato cells were detected no earlier than 7 dpi.We detected no cells dually labeled with Venus and tdTomato after infection.We used this system to assess infection of several organs at 2 dpi as well as at 20 and 60 dpi, when infectious virus was no longer detected.For both experimental systems, we analyzed at least 5 mice and 10-20 slides per mouse.We found virus predominantly, if not solely, in the respiratory tract of all mice, including the lungs and nasal cavity (Figure 1G).Analysis of mice at 2 dpi showed occasional virus-infected cells in the intestine but not in the brain, heart, spleen, or liver (Supplemental Table 2).The intestine was the only extrapulmonary organ in which infected cells could be detected at 2 dpi (Figure 1G and Supplemental Table 2).In the intestine, a common site of SARS-CoV-2 infection in patients (6), virus was detected in occasional, isolated cells, suggesting that infection was nonproductive or, if productive, spread to adjacent cells was inefficient.